Cholera toxin (CT) is an AB5 toxin that consists of a catalytic A1 subunit, an A2 linker, and a cell-binding B pentamer. The separation of CTA1 from CTA2/CTB5 is required for in vivo toxin activity. This occurs after the holotoxin travels by vesicle carriers from the plasma membrane to the endoplasmic reticulum (ER) of a target cell. Reduction of the CTA1/CTA2 disulfide bond occurs at the resident redox state of the ER, but the reduced toxin remains intact. CTA1 must be actively displaced from its non-covalent assembly in the reduced holotoxin by protein disulfide isomerase (PDI), an ER-localized protein with linked but distinct functions as a chaperone and oxidoreductase. The free A1 subunit then moves from the ER to the cytosol where it initiates the cellular events leading to a profuse watery diarrhea that causes 1-4 million illnesses and 100,000 deaths per year. The goal of this project is to define the molecular details of an essential but poorly understood event in cholera intoxication: PDI-mediated holotoxin disassembly. Our recent biophysical analysis has provided the foundation to understand this process. We have shown by isotope-edited Fourier transform infrared (FTIR) spectroscopy that PDI unfolds upon contact with CTA1. A real-time holotoxin disassembly assay demonstrated the displacement of reduced CTA1 from CTA2/CTB5 does not occur when PDI is locked in a folded conformation or when its chaperone function is disrupted by drug treatment. In contrast, the oxidoreductase activity of PDI is not required for CT disassembly. The partial unfolding of PDI provides a molecular basis for CT disassembly: the expanded hydrodynamic size of unfolded PDI would push against two components of the CT holotoxin, thus acting as a wedge to dislodge reduced CTA1 from the rest of the toxin. This phenomenon could also apply to PDI interactions with other AB toxins, and it provides a basis for the established but structurally uncharacterized neuroprotective chaperone activity of PDI: by unfolding in the presence of an amyloid-forming substrate, PDI would act as a ?disaggregase? to displace individual proteins from the neurotoxic aggregate. PDI has an abb'xa' organization that consists of two thioredoxin-like catalytic domains (a & a') separated by two non-catalytic domains (b & b') and an x linker. Based on preliminary and published data, we predict CTA1 binding to the b domain of PDI transmits a signal through the b'x domains for unfolding of the a' domain. We further predict that PDI binds to a region of CTA1 that positions its a' domain near the interface of CTA1 and CTA2. Unfolding of the a' domain would then create a wedge between CTA1 and CTA2, leading to the release of CTA1 from its reduced holotoxin. Interrogation of this model will provide detailed mechanistic insight into the unique and previously unrecognized ?disaggregase? activity of PDI that is responsible for CT disassembly, with potentially broad relevance to toxin biology, neurobiology, and the cell biology of molecular chaperones.